The yeast mitochondrial RNA polymerase has served as a useful model for the study of organellar genome expression. This simple enzyme has just two nuclear encoded subunits, a catalytic core related to bacteriophage enzymes, and a promoter recognition factor with properties similar to bacterial sigma factors. Recent studies have shown that most eukaryotes, including humans, have a gene encoding a core RNA polymerase similar to the one found in yeast. To understand more about the mechanisms of mitochondrial transcription and its regulation we propose to analyze the properties of the wild type and mutant forms of the yeast enzyme. In particular we will determine the specific regions of the two subunits required for protein/protein interactions, and we will analyze domains of the promoter recognition factor for their role in DNA binding, transcription initiation, and release from the elongating RNA polymerase. Using the recently identified clone for the human core polymerase we will isolate and characterize the gene for the human homologue of the promoter recognition factor. Comparison of these human genes to those found in fungi and other eukaryotes will aid our determination of regions of the proteins essential for function, and will help to elucidate the currently unknown mechanisms of mammalian mitochondrial transcription. Little is known about how mitochondrial transcription is regulated. In yeast, transcription of mitochondrial genes varies in response to carbon source. We have found that regulation requires nuclear encoded factors in addition to the subunits of the mitochondrial RNA polymerase. We will study the pathway of regulation from the nucleus to the mitochondrion to determine how these factors modulate the RNA polymerase activity. The proposed studies will aid our understanding of the coordination of nuclear and mitochondrial gene expression, and will help to create a model for the study of human diseases caused by aberrant expression of mitochondrial genes.